Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.
The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region.
Laurberg
M
M
Lund University, Molecular Biophysics, Box 124, S-22100 Lund, Sweden.
Mansilla
F
F
Clark
B F
BF
Knudsen
C R
CR
D004926:Escherichia coli / Q000235:genetics; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu